This study investigates the analytical characterization of full, intermediate, and empty adeno-associated virus (AAV) capsids. AAV vectors are produced through transient transfection of HEK293 cells, resulting in three types of capsids: full, intermediate, and empty. While full capsids are the intended product, intermediate and empty capsids are considered impurities. The presence of these impurities can affect the efficacy and safety of AAV products due to potential competition with full capsids for cellular transduction and increased capsid load during dosing. To assess the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro and in vivo potency assays, the study found that intermediate capsids contribute to the vector genome titer and are equally infectious as full capsids, but do not contribute to the potency of the AAV product. This study confirms the importance of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product. The study used various analytical methods, including analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), and next-generation sequencing (NGS), to characterize the capsids and their impact on potency. Results showed that intermediate capsids contain predominantly snapback partial vector genomes and are not functional in in vitro and in vivo potency assays. The study also demonstrated that the presence of full capsids is critical for potency, as intermediate capsids do not contribute to the relative gene expression. The findings highlight the importance of fully characterizing AAV vectors at the capsid, genome, and functional levels to ensure the best quality vector is delivered to patients. The study confirms the criticality of achieving a high percentage of full vector to generate a safe and efficacious AAV product. Improved vector quality can be achieved through optimization of vector design and bioreactor processes to reduce the biosynthetic generation of intermediate AAV capsids and improvement of the purification process to reduce empty capsids.This study investigates the analytical characterization of full, intermediate, and empty adeno-associated virus (AAV) capsids. AAV vectors are produced through transient transfection of HEK293 cells, resulting in three types of capsids: full, intermediate, and empty. While full capsids are the intended product, intermediate and empty capsids are considered impurities. The presence of these impurities can affect the efficacy and safety of AAV products due to potential competition with full capsids for cellular transduction and increased capsid load during dosing. To assess the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro and in vivo potency assays, the study found that intermediate capsids contribute to the vector genome titer and are equally infectious as full capsids, but do not contribute to the potency of the AAV product. This study confirms the importance of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product. The study used various analytical methods, including analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), and next-generation sequencing (NGS), to characterize the capsids and their impact on potency. Results showed that intermediate capsids contain predominantly snapback partial vector genomes and are not functional in in vitro and in vivo potency assays. The study also demonstrated that the presence of full capsids is critical for potency, as intermediate capsids do not contribute to the relative gene expression. The findings highlight the importance of fully characterizing AAV vectors at the capsid, genome, and functional levels to ensure the best quality vector is delivered to patients. The study confirms the criticality of achieving a high percentage of full vector to generate a safe and efficacious AAV product. Improved vector quality can be achieved through optimization of vector design and bioreactor processes to reduce the biosynthetic generation of intermediate AAV capsids and improvement of the purification process to reduce empty capsids.